Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for rapid cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce normal cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We previously established that the topography of the extracellular matrix (ECM) plays a vital role in regulating cytoskeletal organization, cell morphology, and cell migration by demonstrating that one-dimensional (1D) micropatterned lines mimic the functions of the fibrillar ECM structures found in three-dimensional cell-derived matrix. We have extended these studies to establish the mechanism by which fibrillar topography evokes rapid, efficient cell migration in fibroblasts and how this mode of migration differs from migration studied previously using regular two-dimensional (2D) tissue culture substrates. We found that two key processes of mesenchymal cell migration, protrusion of the leading edge and adhesions formed within the lamella, are enhanced during 1D migration and are controlled indirectly by cellular contractility. Using high-speed imaging, we discovered that protrusions of the cell membrane at the leading edge of migrating cells form more rapidly and frequently during 1D migration. This enhancement depends on reducing lateral adhesion area to a very narrow substrate (1.5 micrometers wide). Quantification showed 2.8- and 10-fold increases in leading edge protrusion rate and net protrusion, respectively, with a 50% increase in migration rate. In contrast, 2D migration is characterized by a wide lamellipodium at the leading edge with multiple cell adhesions spread laterally along the lamellipodium-lamellum border. These findings indicate that physical ECM constraints can regulate leading edge dynamics, adhesions, and the speed of cell migration. Unexpectedly, both 1D adhesions and 3D adhesions to cell-derived matrix fibrils were found to remain more stably associated with the matrix to a substantial degree (e.g., 6-fold longer than in 2D). To examine protein dynamics within adhesions, we used fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoconversion (FLAP) experiments with cells expressing GFP-tagged or KikGR-tagged adhesion molecules. Kinetic analysis revealed that paxillin, vinculin, and actin within 1D adhesions remain more stably associated with the underlying matrix compared to adhesions formed on 2D substrates, consistent with prolonged cell adhesiveness. Interestingly, while integrins on 2D substrates demonstrated retrograde flow towards the cell center, integrins within 1D adhesions remained stable with respect to the ECM, suggesting increased engagement of the physical link between the ECM and the actin cytoskeleton. Loss of contractility after inhibiting myosin II function with blebbistatin or conditional knockout of myosin IIA disrupted 1D adhesion stability and reduced leading edge protrusion efficiency. We suggest that myosin IIA increases cell adhesiveness to fibrillar substrates by stabilizing adhesion proteins found within a molecular clutch between the cell and the substrate, which in turn regulates migration efficiency. We are continuing to explore the role of ECM topography in regulating fibroblast adhesion, migration, and mechanotransduction using 3D collagen hydrogels to compare with 3D cell-derived matrix. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosins and microtubules. All of these processes need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.